The invention relates to novel calpain-like protease nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
Calpains refer to calcium-activated neutral proteinases, a superfamily of endopeptidases typically having cysteine-proteinase and calcium-binding characteristics. These proteinases cleave numerous substrate proteins in a limited manner, typically leading to modification of the function and/or activity rather than general degradation of the substrate.
Calpains are classified into two main groups, the typical or conventional calpains and the atypical calpains, based on their domain content and/or variation. The typical calpains are further subdivided into ubiquitous and tissue-specific calpains based on their predominate patterns of expression.
Two forms of ubiquitous calpains have been extensively characterized in vertebrates: the xcexc-calpains (calpain I, CAPN1) and the m-calpains (calpain II, CAPN2), which are activated in vitro by micro- and millimolar calcium concentrations, respectively. An intermediate xcexc/m calpain has been characterized in chicken.
The ubiquitous xcexc- and m-calpains are heterodimers, each having a distinct, but homologous, large 80 kDa subunit (referred to as xcexcCL or mCL, respectively) and an identical small 30 kDa subunit (referred to as 30K or Cs). The large subunit has four domains, designated I-IV from the N-terminus to the C-terminus. The function of domain I is unclear. Domain II is the cysteine protease domain responsible for calpain protease activity. Domain III is homologous to a calmodulin-binding protein and is speculated to interact with the calcium-binding domains of the large (domain IV) and small subunits (domain VI), when calcium is bound, thereby freeing the protease domain for activity (Goll et al. (1992) BioEssays 14:549-556). Domain IV of the large subunit is a calmodulin-like calcium-binding domain containing four EF-hand calcium-binding motifs. Although structurally similar to calmodulin, domain IV is more similar to sorcin, ALG-2, and grancalcin. Sorcin is involved in the multi-drug resistance of cultured cell lines and was recently reported to associate with the cardiac ryanodine receptor. Grancalcin possibly plays a role in granule-membrane fusion and degranulation. ALG-2 is thought to be involved in apoptosis and is induced by tumor promoters. See Meyers et al. (1995) J. Biol. Chem. 270:26411-26418; Meyers et al. (1985) J. Cell Biol. 100:588-597, Vito et al. (1996) Science 271:521-525; Teahan et al. (1992) Biochem. J. 286:549-554; Boyhan et al. (1992) J. Biol. Chem. 267:2928-2933.
The large subunit of calpains is the catalytic subunit. Three non-contiguous amino acid residues, Cys, His, and Asn, residing within domain II are part of the active site. A recombinant calpain consisting essentially of domains I, II, and III showed calcium-independent activity. Thus, it has been concluded that domain II, but not IV, is necessary and sufficient for protease activity. See Vilei et al. (1997) J. Biol. Chem. 272:25802-25808; and Suzuki et al. (1998) FEBS Letters 433(1, 2):1-4.
The small subunit of typical calpains contains two domains, which are designated V and VI from the N-terminus to the C-terminus. Domain V is an N-terminal glycine-clustering hydrophobic region. Domain VI, which is similar to domain IV of the large subunit, is also a calcium-binding domain containing six EF-hands, EF2-EF5 as in the large subunit, and EF1 and EF6. EF5 of domain VI does not bind calcium and is proposed to be involved in the heterodimeric binding of domains IV and VI during interaction between the large and small subunits.
Not all calpains contain a small subunit, which is identified as a regulator of calpain activity by acting as an inhibitor or pseudosubstrate. In heterodimeric calpains, the small subunit may regulate the calcium-sensitivity of calpain by association and dissociation (Yoshizawa et al. (1995) Biochem. Biophys. Res. Commun. 208:376-383). However, the subunits remain associated during catalysis (Zhang et at. (1996) Biochem. Biophys. Res. Commun. 227:890-896).
The mechanism of activation of calpains is not entirely clear. Suggested mechanisms include combinations of N-terminal autolysis of subunits, homo- and heterodimer association/dissociation, the ratio and binding status of calpains to the calpain endogenous inhibitor calpastatin, calcium presence and concentration, and the redox state of the active site. See Johnson et al. (1997) BioEssays 19(11):1011-1018.
Because xcexc- and m-calpain are activated by in vitro calcium concentrations significantly above physiological levels, in vivo mechanisms that lower the calcium requirement have been proposed. Such mechanisms include interactions with membrane phospholipids and/or membrane associated proteins. See Inomata et al. (1990) Biochem. Biophys. Res. Comm. 171:625-632; and Inomata et al. (1995) Biochim. Biophys. Acta. 1235:107-114.
An activator protein specific for rat brain xcexc-calpain has been isolated and sequenced by Melloni et al. (1998) J. Biol. Chem. 273:12827-12831. Another activator protein specific for m-calpain is found in skeletal muscle. In addition, phospholipids, especially acidic phospholipids, have been found to greatly reduce the calcium concentration necessary for activation. Other activators and factors including DNA have been reported (Mellgren (1991) J. Biol. Chem. 266:13920-13924).
Calpastatin is an endogenous inhibitor of most calpains, the tissue-specific calpain p94 being an exception. Calpastatin, which has five domains, is cleaved by calpain in the interdomain regions, generating inhibitory peptides. The inhibitory effect of calpastatin has been attributed to interactions with calpain domains II, III, IV, and VI. The reactive site of calpastatin shows no apparent homology to that of other protease inhibitors, and it contains the consensus sequence TIPPXYR (SEQ ID NO:6), which is essential for inhibition. See Kawasaki et al. (1989) J. Biochem. 106:274-281, Croall et al. (1994) Biochem. 33:13223-13230; Croall et al. (1991) Physiol. Rev. 71:813-847; Kawasaki et al. (1996) Mol. Membr. Biol. 13:217-224; Melloni et al. (1989) Trends Neurosci. 12:438-444; Sorimachi et al. (1997) J. Biochem. 328:721-732; and Johnson et al. (1997) BioEssays 19(11):1011-1018.
Synthetic active-site inhibitors with varying specificities for calpain and other cysteine proteases include E-64 and derivatives of E-64; leupeptin (N-acetyl-Leu-Leu-argininal); calpain inhibitors I (N-acetyl-Leu-Leu-norleucinal) and II (N-acetyl-Leu-Leu-methioninal); oxoamide inhibitor molecules AK295, AK275, and CX275; and derivatives of peptidyl xcex1-oxo compounds. In contrast to these active-site inhibitors, PD150606 inhibits calpains by binding the calcium-binding domains. The combination of PD150606 and an active site inhibitor such as AK295 can inhibit calpain with high specificity. See Figueiredo-Pereira et al. (1994) J. Neuro. Chem. 62:1989-1994); Tsubuki et al. (1996) J. Biochem. (Tokyo) 119:572-576); and Sorimachi et al. (1997) J. Biochem. 328:721-732. Wang et al (1997) Advances in Pharmacology, Volume 37.
Several typical tissue-specific calpains are known in vertebrates, including skeletal muscle p94 (nCL-1, calpain 3xe2x80x2, CAPN3), stomach nCL2 (CAPN4) and nCL 2xe2x80x2, and digestive tubule nCL4. While p94 contains EF hands, it does not require calcium for proteinase activity. p94 has a domain IV sequence similar to that of xcexcCL and mCL, but it does not bind to a small 30 kDa subunit (Kinbara et al. (1997) Arch. Biochem. Biophys. 342:99-107). p94 contains unique insertion sequences called IS1 and IS2, which are found in domain II and between domains III and IV, respectively). IS2 contains a nuclear-localization-signal-like basic sequence (Arg-Pro-Xaa-Lys-Lys-Lys-Lys-x-Lys-Pro). Connectin/titin binding is also attributed to IS2. p94 may change its localization in a cell-cycle dependent manner and may be involved in muscle differentiation by interacting with the MyoD family. In fact, a defect in the protease p94 is responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). See Sorimachi et al. (1995) J. Biol. Chem. 270:31158-31162; Sorimachi et al. (1993) J. Biol. Chem. 268:10593-10605; Gregoriou et al. (1994) Eur. J. Biochem. 223:455-464; and Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2):135-145.
Atypical calpains include the fungal protein PalB, the yeast PalB homolog, the Caenorhabditis elegans protein Tra-3, human CAPN5 (htra3), CAPN6, and murine CAPN7. Although atypical calpains have a cysteine protease domain homologous to domain II of the large subunit of typical calpains, they lack a calcium-binding domain in the C-terminal portion of the protein (domain IV). See Suzuki et al. (1998) FEBS Letters 433(1, 2):1-4; Sorimachi et al. (1997) J. Biochem. 328:721-732; Franz et al. (1999) Mammalian Genome 10(3):318-321; Goll et al. (1992) BioEssays 14:549-556; and Lin et al. (1997) Nature Struct. Biol. 4:539-547.
PalB, which is involved in the alkaline adaptation of Aspergillus nidulans, is unusual in that it only has a cysteine protease domain. Tra3, which is involved in the sex-determination cascade during early development, has domains similar to domains I, II, and III of the typical calpain large subunit. Human and mouse Tra3 homologs have been identified and localized to x chromosomes, suggesting a role for calpain in sex determination in mammals. See Barnes et al. (1996) EMBO J. 15:44774484; and Sorimachi et al. (1997) J. Biochem. 328:721-732.
The atypical mammalian calpains include CAPN5, 6, and 7. CAPN6 and 7 contain distinct T domains in their C-terminal regions and may not associate with small subunits. These T domains have no significant homology to the calmodulin-like calcium-binding C-terminal domain of other calpains. Furthermore, CAPN6 lacks residues believed to be critical for the active site and may lack protease activity. See Franz et al. (1999) Mammalian Genome 10(3):318-321.
Calpains have broad physiological and pathological roles related to the enzymes"" diverse population of substrates. Calpain substrates include xe2x80x9cPESTxe2x80x9d proteins, which have high proline, glutamine, serine, and threonine contents; calpain and calpastatin; signal transduction proteins including protein kinase C, transcription factors c-Jun, c-Fos, and xcex1-subunit of heterotrimeric G proteins; proteins involved in cell proliferation and cancer including P53 tumor suppressor, growth factor receptors (e.g., epidermal growth factor receptor), c-Jun, c-Fos, and N-myc; proteins with established physiological roles in muscle including Ca++-ATPase, Band III, troponin, tropomyosin, and myosin light chain kinase; myotonin protein kinase; proteins with established physiological roles in the brain and the central nervous system including myelin proteins, myelin basic protein (MBP), axonal neurofilament protein (NFP), myelin protein MAG; cytosketetal and cell adhesion proteins including troponins, talin, neurofilaments, spectrin, microtubule associated protein MAP-2, tau, MAPIB, fodrin, desmin, xcex1-actinin, vimentin, spectrin, integrin, cadherin, filamin, and N-CAM; enzymes including protein kinases A and C, and phospholipase C; and histones. See Sorimachi et al. (1997) J. Biochem. 328:721-732; Johnson et al. (1997) BioEssays 19(11): 1011-1018; Shields et al. (1999) J. Neuroscience Res. 55(5):533-541; and Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2): 135-145.
Substrates of calpain have been associated into several classes including cytoskeletal and structural proteins, membrane bound receptors and proteins, calmodulin binding proteins, enzymes myofibrillar proteins and transcription factors. The examples of the first group include spectrin, MAP-2a, tau factor, neurofilament H, M and L, xcex1-actinin. Examples of the second class include EGF receptor, AMPA-receptor, calcium pump, anion channel, calcium release channel, L-type calcium channel, G-proteins. Examples of the third class include calcium pump, calcineurin, CaM-dependent protein kinase II, myosin light chain kinase, neuromodulin, connexins, IP3 kinase. Examples of the fourth group include protein kinase C, HMG-CoA reductase, cAMP-dependent kinase, pyruvate kinase, phosphorylase kinase. Examples of the fifth group include troponin I, troponin T, tropomyosin, myosin.. Examples of the sixth group include c-fos, c-jun, Pit-1, Oct-1, and b, c-Myc. See Wang, et al. (1997) Advances in Pharmacology, Volume 37).
Calpain is implicated in a wide variety of physiological processes including alteration of membrane morphology, long-term potentiation of memory, axonal regeneration, neurite extension, cell proliferation (division), gastric HCl secretion, embryonic development, secretory granule movement, cell differentiation and regulation, cytoskeletal and membrane changes during cell migration, cytoskeletal remodeling, sex determination, and alkaline adaptation in fungi. See Solary et al. (1998) Cell Biol. Toxicol. 14:121-132; Sorimachi et al. (1997) J. Biochem. 328:721-732, Johnson et al. (1997) BioEssays 19(11):1011-1018; Suzuki et al. (1998) FEBS Letters 433(1, 2):1-4, Franz et al. (1999) Mammalian Genome 10(3):318-321; Shields et al. (1999) J. Neuroscience Res. 55(5):533-541, Schnellmann et al. (1998) Renal Failure 20(5):679-686; Banik et al. (1998) Annals New York Acad. Sci. 844:131-137; Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2):135-145; and McIntosh et al. (1998) J. Neurotrauma 15(10):731-769.
Under pathological conditions, aberrant regulation and/or activity of calpain can be detrimental to cells and tissues. In this context, calpains are implicated in a wide variety of disease states including exercise-induced injury and repair; apoptosis including T cell receptor-induced apoptosis, HIV-infected cell apoptosis, ectoposide-treated cell apoptosis, nerve growth factor deprived neuronal apoptosis; ischemia, such as cerebral and myocardial ischemia, traumatic brain injury; Alzheimer""s disease and other neurodegenerative diseases; demyelinating diseases including experimental allergic encephalomyelitis (EAE) and multiple sclerosis; LGMD2A muscular dystrophy; spinal cord injury (SCI); cancer; cataract formation; and renal cell death by diverse toxicants.
Optic nerve degeneration is one of the most common features of optic neuritis that leads to impaired vision and possible blindness. This condition is one of the first manifestations of multiple sclerosis. The mechanism of optic nerve degeneration in optic neuritis has been studied in experimental allergic encephalomyelitis, an animal model of optic neuritis. Calpain is present in the central nervous system and degrades myelin proteins. The role of calpain in demyelination associated with optic neuritis has been evaluated in rats with experimental optic neuritis. The results show that increased activity and translational expression of calpain in optic neuritis may be integral to the pathogenesis of this disorder. See Banik et al. (1999) Histol Histopathol 14: 649-656. The pathophysiological role of calpain in experimental demyelination has also been reviewed in Shields et al. (1999) Journal of Neuroscience Research 55:533-541.
Abnormality of protease activities and the imbalance of intracellular calcium are two key defects in Alzheimer""s disease. Accordingly, it has been suggested that calcium dependent proteases such as calpain, as a critical link between these two events, must play a key role in the pathogenesis of Alzheimer""s disease, and especially in abnormal processing of beta-amyloid precursor protein. See Chen, et al., (1998) Frontiers in Bioscience 3, a66-75. Further, calpains have been implicated in renal cell death. Schnellmann et al. ((1998) Renal Failure, 20(5): 679-686) showed that the inhibition of calpain activity decreased cell death produced by various toxicants. The role of calpain has also been studied in exercise-induced muscle injury. See Belcastro et al. (1998) Molecular and Cellular Biochemistry 179:135-145. The role of calpain homologs has been reviewed by Sorimachi et al. (1997) Biochem, J. 328:721-732. For example, one mammalian homolog, predominantly expressed in skeletal muscle has shown to be responsible for limb girdle muscular dystrophy type 2a. Another calpain homolog in nematodes is involved in the sex determination cascade during early development. Such calpain homologs in mammals have been found to be predominantly expressed in a limited number of organs in contrast with the ubiquitous expression of the more common calpains. These tissue-specific calpains include skeletal muscle-specific and stomach-specific. These homologs contain a cysteine-protease domain showing similarity to the more common calpain large subunit than to other cysteine proteases. However, they are atypical in that their other domains do not necessarily resemble conventional calpain large subunits. These have been reviewed in Sorimachi et al., above. Calcium isoforms in subunits are shown in, for example, Wang et al.(1997) Advances in Pharmacology 37:117-152.
Evidence has also suggested that calpains play a role in the pathology of cerebral ischemia. See Zalewska (1996) Folia Neuropathol. 34.3. Suppressive and protective effects of calpain inhibitors has been shown on post-ischemic damage. This subject has been reviewed in Zalewska. Silver et al. (1996) Clinical Neuropharmacology 19:101-128 has reviewed various medical therapies for ischemic stroke. This includes cytoprotective therapy with drugs that prevent ischemia and reperfusion including calpain inhibitors.
Given the diversity of calpains in cellular processes and disease states, compositions and methods directed to calpains are useful to influence calpain activity in a variety of tissues, thereby extending protection to cells and tissues affected with aberrant calpain function and/or regulation.
Isolated nucleic acid molecules corresponding to calpain-like protease nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2 or the nucleotide sequences encoding the DNA sequence deposited in a bacterial host with the ATCC as Patent Deposit Number PTA-2203. Further provided are calpain-like protease polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
Another aspect of this invention features isolated or recombinant calpain-like protease proteins and polypeptides. Preferred calpain-like protease proteins and polypeptides possess at least one biological activity possessed by naturally occurring calpain-like protease proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listing are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the calpain-like protease polypeptides and fragments are provided. Such antibodies are useful in detecting the calpain-like protease polypeptides as well as in regulating the T-cell immune response and cellular activity, particularly growth and proliferation.
In another aspect, the present invention provides a method for detecting the presence of calpain-like protease activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of calpain-like protease activity such that the presence of calpain-like protease activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating calpain-like protease activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) calpain-like protease activity or expression such that calpain-like protease activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to calpain-like protease protein. In another embodiment, the agent modulates expression of calpain-like protease protein by modulating transcription of a calpain-like protease gene, splicing of a calpain-like protease mRNA, or translation of a calpain-like protease mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the calpain-like protease mRNA or the calpain-like protease gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant calpain-like protease protein activity or nucleic acid expression by administering an agent that is a calpain-like protease modulator to the subject. In one embodiment, the calpain-like protease modulator is a calpain-like protease protein. In another embodiment, the calpain-like protease modulator is a calpain-like protease nucleic acid molecule. In other embodiments, the calpain-like protease modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding a calpain-like protease protein; (2) misregulation of a gene encoding a calpain-like protease protein; and (3) aberrant post-translational modification of a calpain-like protease protein, wherein a wild-type form of the gene encodes a protein with a calpain-like protease activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a calpain-like protease protein. In general, such methods entail measuring a biological activity of a calpain-like protease protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the calpain-like protease protein.
The invention also features methods for identifying a compound that modulates the expression of calpain-like protease genes by measuring the expression of the calpain-like protease sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.